Two New Cytotoxic Sesquiterpene-Amino Acid Conjugates and a Coumarin-Glucoside from Crossostephium chinense

The Asteraceae family is a promising source of bioactive compounds, such as the famous Asteraceae plants Tanacetum cinerariifolium (pyrethrin) and Artemisia annua (artemisinin). As a result of our series of phytochemical studies of the subtropical plants, two novel sesquiterpenes, named crossoseamines A and B in this study (1 and 2, respectively), one undescribed coumarin-glucoside (3), and eighteen known compounds (4–21) were isolated from the aerial part of Crossostephium chinense (Asteraceae). The structures of isolated compounds were elucidated by spectroscopic methods, including 1D and 2D NMR experiments (1H, 13C, DEPT, COSY, HSQC, HMBC, and NOESY), IR spectrum, circular dichroism spectrum (CD), and high-resolution electrospray ionization–mass spectrometry (HR-ESI–MS). All isolated compounds were evaluated for their cytotoxic activities against Leishmania major, Plasmodium falciparum, Trypanosoma brucei (gambiense and rhodesiense), and human lung cancer cell line A549 because of the high demand for the discovery of new drug leads to overcome the present side effects and emerging drug-resistant strains. As a result, the new compounds (1 and 2) showed significant activities against A549 (IC50, 1: 3.3 ± 0.3; 2: 12.3 ± 1.0 μg/mL), L. major (IC50, 1: 6.9 ± 0.6; 2: 24.9 ± 2.2 μg/mL), and P. falciparum (IC50, 1: 12.1 ± 1.1; 2: 15.6 ± 1.2 μg/mL).


Introduction
Crossostephium chinense (Asteraceae) is an evergreen shrub distributed in Japan, China, and other Asian countries. The shrub naturally grows on coastal raised coral reefs and can reach 10-40 cm in height. From November to December, the racemes are borne on the terminal branches and bloom tiny yellow flowers. C. chinense is widely planted as an ornamental shrub in China because of the high rate of survival for artificial cultivation [1]. The whole plants of C. chinense are traditionally used for the treatment of diabetes and arthritis in southward China [2]. The extract has been reported to have various activities, such as preventing the progression of restenosis [3], inhibiting differentiation, formation and bone resorptive abilities of osteoclasts [4], and larvicidal activity [5]. Flavonoids [6,7], coumarin [2,8], sesquiterpene [2], and triterpenoid [9] have previously been isolated from C. chinense; however, the phytochemical analysis is still insufficient, and a detailed evaluation of biological activities, especially for anticancer and antiparasitic activities, has yet to be conducted.

Plausible Biosynthetic Pathway of 1 and 2
The plausible biosynthetic pathway of crossoseamines A and B (1 and 2) is the Michaeltype reaction of the nitrogen of proline to the eudesmanolide moiety (Figure 7). Yoshikawa et al. (1993) have reported a chemical synthesis of similar proline conjugates by the reaction of α-methylene γ-lactone type eudesmanolide and proline with Et 3 N at 90 • C [50]. The demand for a relatively strong base and high temperature for chemical synthesis indicates the involvement of the biosynthetic enzyme to form 1 and 2 in C. chinense, not as artifacts through the extraction and purification processes. The 6,7-dihydroxy coumarin moiety of 3 is thought to be the lactone form of caffeic acid, i.e., esculetin, which undergoes further methylation, glycosylation, and caffeoylation to 3. Given the other isolated compounds having caffeoyl function, C. chinense is considered a rich source of caffeic acid and its derivatives.
Molecules 2023, 28, 4696 8 of 15 [50]. The demand for a relatively strong base and high temperature for chemical synthesis indicates the involvement of the biosynthetic enzyme to form 1 and 2 in C. chinense, not as artifacts through the extraction and purification processes. The 6,7-dihydroxy coumarin moiety of 3 is thought to be the lactone form of caffeic acid, i.e., esculetin, which undergoes further methylation, glycosylation, and caffeoylation to 3. Given the other isolated compounds having caffeoyl function, C. chinense is considered a rich source of caffeic acid and its derivatives.

Cytotoxic Activities of the Isolated Compounds
All isolated compounds from the EtOAc fraction of C. chinense were evaluated for their cytotoxic activities against L. major, P. falciparum, T. brucei (gambiense and rhodesiense), and human lung cancer cell line A549 (Table 3).
The cytotoxicity against the human lung cancer cell line, A549, and the pathogen of cutaneous leishmaniasis, L. major, were evaluated by MTT method with positive control etoposide and miltefosine, respectively. The results are summarized in Table 3. Compounds 1 and 2 and some flavonoids (6-8) showed significant activities comparable to the positive controls.
The anti-Plasmodium activity was evaluated by the SYBR Green I method. The activity of compounds 1, 2, 11, and 14 are reported for the first time, whereas that of compounds 6-9, 12, 13, 15, and 18 was previously reported [37][38][39][40]. The reported activities of the above compounds were consistent with our results, with some deviations (Table S2). The slight differences were ascribable to the differences in the assay methods and conditions and species/strains of the parasites used.

Cytotoxic Activities of the Isolated Compounds
All isolated compounds from the EtOAc fraction of C. chinense were evaluated for their cytotoxic activities against L. major, P. falciparum, T. brucei (gambiense and rhodesiense), and human lung cancer cell line A549 (Table 3). The cytotoxicity against the human lung cancer cell line, A549, and the pathogen of cutaneous leishmaniasis, L. major, were evaluated by MTT method with positive control etoposide and miltefosine, respectively. The results are summarized in Table 3. Compounds 1 and 2 and some flavonoids (6)(7)(8) showed significant activities comparable to the positive controls.
The anti-Plasmodium activity was evaluated by the SYBR Green I method. The activity of compounds 1, 2, 11, and 14 are reported for the first time, whereas that of compounds 6-9, 12, 13, 15, and 18 was previously reported [37][38][39][40]. The reported activities of the above compounds were consistent with our results, with some deviations (Table S2). The slight differences were ascribable to the differences in the assay methods and conditions and species/strains of the parasites used.

General Experimental Procedure
Optical rotations were measured on a P-1030 spectropolarimeter (JASCO, Tokyo, Japan). IR and UV spectra were measured on FT-720 (HORIBA, Kyoto, Japan) and V-520 UV/Vis spectrophotometers (JASCO, Japan), respectively. 1 H-and 13 C-NMR spectra were measured on Avance III HD spectrometer (Bruker, Billerica, MA, USA) at 500 and 125 MHz, respectively, with the residual solvent signal as references. Positive-and negative-ion HR-ESI-MSs were recorded on an LTQ Orbitrap XL spectrometer (Thermo Fisher Scientific, Waltham, MA, USA), and MS/MS fragments of precursor ions were detected by the CID mode with a collision energy of 35 eV.

Plant Material
The aerial parts of C. chinense were collected in July 2008 in Okinawa, Japan, and a voucher specimen was deposited in the Herbarium of the Department of Pharmacognosy, Graduate School of Biomedical Sciences, Hiroshima (deposition number: 08-CC-Okinawa-0708).

Extraction and Isolation
Air-dried aerial parts of C. chinense (3.5 kg) were extracted with MeOH (3 × 10 L) at room temperature. The methanol extract was concentrated to 1.5 L and then partitioned with an equal volume of n-hexane to obtain an n-hexane-soluble layer (27.7 g). The remaining layer was evaporated and resuspended in 1.5 L of water and then extracted with 1.5 L of EtOAc and 1.5 L of 1-BuOH successively to obtain EtOAC (74.

Acid Hydrolysis of Compounds 1 and 2
Compounds 1 and 2 (0.2 mg each) were treated with 1% aqueous hydrochloric acid (HCl) (0.5 mL) at room temperature for 12 h. The reaction mixture was extracted with EtOAc to obtain the EtOAc and aqueous layers. The latter was subjected to HPLC analysis with an optical rotation detector (OR-2090 plus; JASCO) on a HILIC column (Cosmosil HILIC, 10 × 250 mm, CH 3 CN-H 2 O (4:1, v/v), flow rate: 0.7 mL/min). The peaks from 1 and 2 were identical with an authentic standard, L-proline (t R : 30.0 min, negative optical rotation) [51].

Sugar Analysis of Compound 3
Compound 3 (0.5 mg) was hydrolyzed with 1M HCl (0.2 mL) at 80 • C for 2 h. After cooling, EtOAc was used to extract the reaction mixture, and the aqueous layer was subjected to HPLC analysis with an optical rotation detector (OR-2090 plus; JASCO) on an amino column [Asahipak NH 2 P-50 4E, 4.6 × 250 mm, CH 3 CN-H 2 O (3:1, v/v), flow rate: 1 mL/min] to identify the D-glucose from 3, which was determined by comparison of its retention time and optical rotation sign with authentic sample (t R : 7.3 min, positive optical rotation).

Growth Inhibition Activity
The evaluation of cytotoxic activities against A549, L. major, P. falciparum, and T. brucei (gambiense and rhodesiense) was conducted following previous reports.
In brief, the human lung cancer cell line A549 was maintained in 10% FCS-supplemented DMEM. Various concentrations of samples in dimethylsulfoxide (DMSO) and A549 (5 × 10 3 cells/well) were cultured in a CO 2 incubator for 72 h. The medium was replaced with 100 µL of MTT solution and incubated for 1.5 h in the same condition. The viability was calculated from the absorbance of formed MTT formazan at 550 nm using a microplate reader [52]. Etoposide was used as a positive control.
The leishmanicidal activities of the isolated compounds were also evaluated using an MTT assay. In a 96-well plate, various concentrations of sample solutions in dimethylsulfoxide (DMSO) and L. major (2 × 10 5 parasite/well) in 100 µL of M199 medium were incubated for 72 h at 25 • C. Then, 100 µL of MTT solution was replaced and incubated overnight. The absorbance of the formazan solution in DMSO was recorded using a microplate reader at 550 nm [11]. Miltefosine was used as a positive control.
The trypanocidal activities of the isolated compounds were performed in 96-well plates with slight modifications [53]. In brief, each well contains 100 µL of parasite culture (1 × 10 4 parasites/well) with serial dilutions of compounds. After incubation for 72 h at 37 • C under 5% CO 2 , 25 µL of CellTiter-GloTM Luminescent Cell Viability Assay reagent (Promega Japan, Tokyo, Japan) was added to evaluate intracellular ATP concentration according to the instruction. Nifurtimox was used as a positive control.
Anti-Plasmodium activity of the isolated compounds was evaluated according to the previous report [54]. In brief, 100 µL of P. falciparum 3D7 parasite culture [55] was plated in a 96-well plate with various concentrations of the compounds. After incubation for 72 h at 37 • C in a humidified chamber under a gas mixture of 90% N 2 , 5% O 2, and 5% CO 2 , the parasitemia was determined by SYBR Green I assay (Lonza Ltd., Basel, Switzerland) with a microplate reader at 485 and 530 nm. Artemisinin dissolved in DMSO was used as a positive control, and DMSO was used as a negative control. Human erythrocytes and plasma were obtained from the Nagasaki Red Cross Blood Center, and their usage was approved by the ethical committee of the Institute of Tropical Medicine, Nagasaki University.
The 50% inhibitory concentration (IC 50 ) values were obtained for each compound by linear regression method.
Crossoseamine A (1) showed potent activity against A549 and L. major, while the introduction of the hydroxy function on position 8 (crossoseamine B (2)) decreased the activity. The polarity and/or functionality of eudesmane moiety may be necessary for the cytotoxicity against these targets. Among the evaluated compounds, chrysosplenol D (6) showed substantial toxicity for all tested organisms, probably by general toxicity, and has already been reported previously [21,25,56]. While some flavonoids (7-9) and caffeic acid derivatives (14) are relatively specific for both Trypanosoma and Plasmodium, compounds (11-13, 15, 18) showed specificity for Plasmodium. As a result, these compounds have the potential to treat human cancer, leishmaniasis, malaria, and trypanosomiasis. However, further chemical modification and mechanism analyses are needed in the future.